This proposal addresses the fundamental mechanisms involved in the polarized trafficking of plasma membrane proteins in Retinal Pigment Epithelium (RPE). Previous research from our laboratory has revealed dramatic differences in the targeting of several apical and basolateral plasma membrane proteins between RPE and other simple epithelia, represented by the prototype kidney cell line MDCK. Basolateral proteins such as the neural cell adhesion molecule (NCAM) and the chaperone for lactate transporters CD147 (EMMPRIN) have a strikingly reversed apical polarity in adult RPE. Experiments in SA1 will study the role of different basolateral sorting signals in polarized transport between Golgi and plasma membrane in RPE and study the role of CD147 in the polarized sorting of neutral amino acid transporters. Experiments in SA2 will explore the role of basolateral sorting adaptors in RPE. We will explore the RPE trafficking phenotype arising from the absence of AP1B, an adaptor found in most other epithelial cells. This phenotype includes alterations in the trafficking of Transferrin Receptors, compared to AP1B+ epithelial cells, and a dramatically increased susceptibility to adenovirus infection due to altered trafficking in virus receptors. Other experiments will characterize the basolateral sorting role of other adaptors present in RPE (APIA, AP3, AP4, GGA1-3), using an RNAi approach, and search for novel adaptors/proteins that may account for the reversed polarity of NCAM and CD147. Experiments in SA3 will address the mechanisms involved in polarized vesicular delivery in RPE. This aim builds on significant technical and conceptual advances in our understanding of the vesicular delivery machinery of MDCK cells. We will characterize the role of svntaxins 1 and 4, microtubules, cdc42 and IQGAP1 in protein sorting by RPE cells. We will explore the hypothesis that some components of the basolateral vesicular trafficking machinery in MDCK cells may operate in reverse in RPE cells. The data obtained will provide fundamental information on how RPE cells organize their polarized trafficking routes. This information is essential to understand RPE's roles in the physiology of the retina and in blinding diseases, and may help in devising improved gene therapy strategies for their treatment.